PROJECT SUMMARY During development and disease, epithelial cells can migrate collectively, with or without undergoing epithelial- mesenchymal transition (EMT), through heterogeneous matrices, enabling fundamental processes such as branching morphogenesis, fibrosis, and tumor invasion. We have shown that extracellular matrix (ECM) properties beyond the current ECM stiffness, such as confinement and past ECM stiffness, can fundamentally alter epithelial responses. Through a collection of projects, combining experiments and simulations, this proposal will reveal new modes collective cell behaviors in matrices of heterogeneous stiffness and topography. We will measure EMT and migration of epithelial cells around defects in a basement membrane (BM)-like matrix, determine 3D invasion due to defect-induced EMT, build a computational model to understand rate-dependent EMT evolution, and pharmacologically disrupt BM degradation. We will also assess whether the epithelial cells can sense deeply into their matrix and alter responses based on distant stiffening of the matrix. In another project, we will investigate how cell sheets migrate in 3D-like confined environments of tunable stiffness and topography. We will connect nuclear shape with cytoskeletal reorganization to understand how cells adapt to distinct stiffnesses of past and present matrices. Outcomes of these projects will enable new fundamental understanding of epithelial cell responses to matrix heterogeneities that have remained unexplored and could reveal novel targets for diseases such as fibrosis and cancer.